In recent years, there have been remarked various physiological functions owned by sphingolipids as a constituent of the cell membrane lipids of eukaryotic organisms, as well as glycerolipids. Sphingolipid ceramide deacylase (SCDase), which acts on this sphingolipid to generate a fatty acid and a lysosphingolipid, is an enzyme that is not only useful in the elucidation of the physiological actions of sphingolipids but also very important in the field of engineering of sphingolipids such as preparation of derivatives of sphingolipids or labeling of sphingolipids.
Conventionally, an enzyme that acts on ceramide to hydrolyze the acid-amide bond between the sphingosine base and the fatty acid has been known as ceramidase (EC 3.5.1.23) [Journal of Biological Chemistry, 241, 3731–3737 (1966); Biochemistry, 8, 1692–1698. (1969); Biochimica et Biophysica Acta, 176, 339–347 (1969); Science, 178, 1100–1102 (1972)]. However, the enzyme is incapable of hydrolyzing the acid-amide bond between the sphingosine base and the fatty acid in the ceramide moiety of a sphingoglycolipid or sphingomyelin.
On the other hand, enzymes produced by microorganisms belonging to the genus Nocardia, the genus Rhodococcus, or the genus Streptomyces [Journal of Biochemistry, 103, 1–4 (1988); Japanese Patent Laid-Open No. Hei 6-78782; Japanese Patent Laid-Open No. Hei 7-107988] are capable of acting on a sphingoglycolipid to hydrolyze the acid-amide bond between the sphingosine base and the fatty acid, thereby generating a lysosphingoglycolipid and a fatty acid. However, all these enzymes have characteristics such that their substrate specificity is so narrow that they cannot act on all the sphingolipids.
In other words, enzymes produced by microorganisms belonging to the genus Nocardia act on what is so-called an “acidic glycolipid” such as GD1a, GM1, GM2 or GM3, but show little or no action on a neutral glycolipid. On the other hand, enzymes produced by microorganisms belonging to the genus Rhodococcus act on a neutral glycolipid but are incapable of acting on an acidic glycolipid. Enzymes produced by microorganisms belonging to the genus Streptomyces do not act on GM3 or a neutral glycolipid such as lactosylceramide or cerebroside. In addition, none of the enzymes described above act on sphingomyelin.
On the other hand, the SCDase derived from Pseudomonas sp. TK-4 strain [Journal of Biological Chemistry, 270, 24370–24374 (1995); Japanese Patent Laid-Open No. Hei 8-84587] has been known to have a broad spectrum of substrate specificity for a general sphingolipid including an acidic glycolipid, a neutral glycolipid, sphingomyelin and the like. In addition, this SCDase catalyzes not only a hydrolytic reaction of a sphingolipid to generate the corresponding lysosphingolipid and the corresponding fatty acid, but also a condensation reaction for synthesizing a sphingolipid from a lysosphingolipid and a fatty acid, and further catalyses a reaction for exchanging a fatty acid moiety of a sphingolipid with another fatty acid (WO 98/03529). Therefore, the SCDase serves as a very important tool in the field of engineering of sphingolipids, and is highly valued for its industrial applications. However, when the SCDase described above is industrially advantageously produced from a microorganism, it is necessary to add a ganglioside mixture to the culture medium during cultivation in order to induce the production of the enzyme existing in nature. For this reason, free fatty acids and lysosphingoglycolipids are produced in the culture medium, and enzymes other than the desired enzyme, such as sphingomyelinase, are concurrently produced, thereby making it difficult to perform the separation and purification of the desired SCDase from these lipids and co-existing enzymes.
In view of the above, in order to produce by genetic engineering the SCDase produced by Pseudomonas sp. TK-4, a gene for this SCDase has been cloned to prepare a transformant in which the gene is introduced (Japanese Patent Laid-Open No. Hei 11-276177). However, the activity of the SCDase produced in the transformant is low or at an undetectable level. Therefore, it has been difficult to produce SCDase by genetic engineering.
An object of the present invention is to provide a polypeptide possessing a sphingolipid ceramide deacylase activity, which is useful in the field of engineering of sphingolipids; a nucleic acid encoding the polypeptide; a production method capable of producing the polypeptide easily and in a large scale by genetic engineering; an oligonucleotide probe or primer, capable of specifically hybridizing to the nucleic acid; a method for detecting a sphingolipid ceramide deacylase gene using the oligonucleotide probe or primer; a kit usable therefor; an antibody or a fragment thereof, capable of specifically binding to the polypeptide of the present invention; a method for detecting a sphingolipid ceramide deacylase using the antibody or a fragment thereof; and a kit usable therefor.